The Isolation of Recombinants between Related Orbiviruses

Transcription

1 J. gen. ViroL (1978), 4I, Printed in Great Britain 333 The Isolation of Recombinants between Related Orbiviruses By B. M. GORMAN, JILL TAYLOR, P. J. WALKER AND P. R. YOUNG Queensland Institute of Medical Research, Bramston Terrace, Herston 4006, Australia* (Accepted I June 1978) SUMMARY Temperature-sensitive mutants of the related orbiviruses, Wallal and Mudjinbarry, recombine with high frequency when grown in pairs in cell culture. The genome of each virus consists of discrete segments of double-stranded RNA and high frequency recombination suggests that reassortment of genome segments occurs rather than classical recombination. Electrophoresis in acrylamide gels of RNA extracted from the progeny of a cross between Wallal ts IOI and Mudjinbarry ts 3 mutants, revealed that three plaque isolates of 6o tested differed in RNA pattern from each of the parent viruses and from each other. Further analysis of the electrophoretic profiles suggested that the isolates were recombinants with RNA segments derived from each of the parent viruses. INTRODUCTION Wallal and Mudjinbarry viruses isolated from Culicoides spp. (Doherty et ai. 1973; Doherty et al. 1977) show characteristic orbivirus structure by electron microscopy (Gorman & Lecatsas, I972; Lecatsas & Gorman, 1972; Doherty et al. 1977). In a comparative study of Wallal and bluetongue viruses, Wallal virus was shown to contain ten segments of doublestranded RNA and seven polypeptides similar in mol. wt. distribution but not identical with those of bluetongue virus (Gorman et al. 1973) Wallal and Mudjinbarry viruses are indistinguishable by complement fixation tests but distinguishable by serum neutralization tests (Doherty et al. 1977) and form part of the Wallal sero-group of orbiviruses. The presence of a segmented genome in orbiviruses suggests that reassortment of genome segments in mixed infections could occur by mechanisms similar to those described for other viruses with segmented genomes, notably influenza virus (Suguira, 1975) and reovirus (Joklik, 1974). Genetic reassortment provides a mechanism for production cf new virus strains differing in one or more RNA segments from the parental types. We report here on high frequency recombination between temperature-sensitive mutants of viruses within an orbivirus serological group and the use of these mutants to produce recombinants with genome segments derived from each of the parent viruses. METHODS Cell cultures. Growth of viruses, plaque assay and plaque reduction neutralization tests were performed in a stable line of pig kidney cells (PS-EK) by methods described previously (Gorman et al. 1975). * Address for reprints: Librarian, Queensland Institute of Medical Research, Bramstort Terrace, Herston 4oo6, Australia. 22 VIR 4X

2 334 B.M. GERMAN AND OTHERS Viruses. Wallal (Chi2o48) and Mudjinbarry (NTI4952) viruses were cloned by plaque technique three times from suckling mouse brain pools. Reovirus type 3 (strain HEV; Stanley, 1961) was obtained after six passages in monkey kidney cells (LLC-MK2) and six passages in mouse fibroblast cells (L-929). Mutagenesis. PS-EK cells infected with wild-type Wallal or Mudjinbarry viruses at a multiplicity of infection of o.oi p.f.u./cell were maintained at 32 C with fluid maintenance medium containing 500/zg/ml of 5-fluorouracil. When infected cultures maintained without 5-flurouracil showed cytopathic effect, all cultures were harvested and tested for yield of mutants. Isolation of temperature-sensitive mutants. Mutagenized virus stocks diluted to give less than ten plaques/culture dish (55 mm diam.) were grown at 32 C. After 4 days the borders of visible plaques were marked and the dishes transferred to 37 C. After 24 h the plates were stained with medium containing o.o1% neutral red and plaques showing no increase in size were isolated as provisional temperature-sensitive mutants. The picked plaques were grown at 32 C in fluid culture and the progeny assayed for p.f.u, at 32 and at 37 C. Suitable cultures were again plaque-cloned twice and tested for temperature sensitivity. Only cultures showing a plaquing efficiency at 37 C of at least ion-fold less than at 32 C were considered temperature-sensitive. Mixed infection. High multiplicity tests were performed in 96-well Linbro microculture plates. Appropriate dilutions of two mutants were added in 0"05 ml amounts to six replicate wells. Six control wells contained only single virus, and the volume was adjusted to o.1 ml with medium I99. All wells then received o-i ml of medium I99 containing i lo 4 cells. Final combined input multiplicity was about 2o p.f.u, per cell. Plates were incubated for 48 h at 32 C, frozert and stored at -7o C. Duplicate samples consisting of three pooled wells each, were centrifuged at 8000 g for 3o min and supernatant fluids assayed for virus plaques at 32 and 37 C. Recombination frequency was calculated from the formula (A x B) 37 C- (A + B) 37 C (Ax B) 32 C x Ioo%, where A and B are the two temperature-sensitive mutants. Low multiplicity tests were performed in monolayer cultures of I x lo 5 cells. Appropriate dilutions of mutants were prepared and inoculated individually to give a combined input multiplicity of 0.02 p.f.u./cell. The multiplicity of a given mutant was the same for both single control cultures and mixed infection. After adsorption of virus at 32 C for I h the inoculum was removed and the cell monolayers washed twice with medium 199. One ml of medium I99 was added to each culture and they were incubated at 32 C until a cytopathic effect occurred. Recombination frequency was determined as for high multiplicity tests. Extraction and p~rification of double-stranded RNA. Virus-infected cell cultures were extracted with sodium dodecyl sulphate and diethyl pyrocarbonate at 37 C (Summers, I97O ). The extract was adjusted to 2 M in lithium chloride and allowed to stand at 4 C for 18 h to precipitate single-stranded RNA (Avital & Elson, 1969). The supernate was applied to a cellulose chromatography column (Whatman CFII) in o.i M-NaC1, o.ooi M-EDTA, o-oi M-tris, ph 7"4 (TSE)+I5% ethanol. Residual single-stranded RNA and DNA was eluted in TSE+ 15 % ethanol and double-stranded RNA in TSE buffer (Franklin, I966). Electrophoresis of RNA. Electrophoresis of RNA in slabs of 7"5 % polyacrylamide gels has been described previously (German et al. 1977).

3 Orbivirus recombinants ,6 i 10 (a) (b) (c) Fig. I Separation of dsrna genome segments of (a) Mudjinbarry virus; (b) a mixture of Wallal and Mudjinbarry viruses; (c) Wallal virus. Electrophoresis was performed in a downward direction in a gel slab of 7"5 % polyacrylamide and RNA stained with methylene blue (Gorman et al. 1977) Table I. Mol. wt. of double-stranded RNA segments of Wallal and Mudjinbarry viruses Segment Wallal M udj in barry I 2"33* 2'33 2 2"16 2'05 3 t "9O I " I'29 5 I'O1 H9 6 I -oi o-51 o-5i 8 o'45 o'45 9 o'4i o'4i IO O'22 O'23 Total 11 "29 I I "34 * Mol. wt. ( Io n) calculated using double-stranded RNA segments of reovirus type 3 as standard (Joklik, 1974). 22-2

4 336 B.M. GORMAN AND OTHERS RESULTS Electrophoretic separation of virus RNA Electrophoresis of double-stranded RNA from Watlal and Mudjinbarry viruses (Fig. I) revealed differences in segments z, 3, 4, 5, 6 and Io. The differences at segments 2, 5 and Io were obvious in comparison of Fig. I (a) and (c) but co-electrophoresis of RNA from both viruses was required (Fig. I b) to detect the small differences in segments 3, 4 and 6. Estimation of tool. wt. of segments by comparison with reovirus RNA segments in the same gels, confirmed the differences between the viruses at segments 2, 5 and Io but the differences in other segments were too small to reveal in this way. The comparative tool. wt. are shown in Table I. Isolation of mutants Wallal and Mudjinbarry wild-type viruses produced 1u-fold to Ioo-fold higher yield when serially passaged at 32 C instead of at 37 C. The efficiency of plating was the same at the two temperatures but plaques produced at 37 C were slightly smaller than those at 32 C. At temperatures higher than 37 C, plaques were extremely small and difficult to count. The non-permissive temperature used was 37 C and the permissive 32 C. From 2298 plaques of Wallal virus which had been grown in the presence of 5-fluorouracil, Iz5 non-enlarging plaques were picked as potentially temperature-sensitive and these yielded 11 stable mutants. From 255o plaques of Mudjinbarry virus which had been grown in the presence of 5-fluorouracil, 1 I3 non-enlarging plaques yielded four stable mutants. Plaque morphology of the mutants did not differ from that of wild-type virus. The efficiency of plating of the mutants is shown in Table 2. Electrophoretic separation of RNA isolated from the mutants did not differ from RNA isolated from the parent viruses. Mixed infections Combinations of the available mutants were used in mixed infection studies to determine at least two recombination groups. Five mutants of Wallal virus and three of Mudjinbarry virus were assigned to two recombinant groups (Table 3). At an input multiplicity of 20 p.f.u./cell recombination occurred between members of different groups and no recombination was found within groups. Mudjinbarry mutants ts 3 and ts I5 recombined with Wallal mutant ts I o I and Mudjinbarry mutant ts 5 with Wallal mutant ts 3o. Recombination frequencies were difficult to establish precisely due to variation in replicate tests. Similar difficulties in establishing recombinatiort frequencies in similar tests using viruses with segmented genomes have been reported (Fields, I971; Suguira, I975; Shipham & De La Rey, I976). Our attempts to standardize frequencies using methods reported for similar tests with reovirus mutants (Fields, I97I) were not successful. High recombination frequencies were also found in mixed infections at low multiplicity (Table 3). Recombination frequencies were again variable but the allocation of mutants to two groups was similar to that established by high multiplicity tests. High frequency recombination in low multiplicity crosses has been demonstrated with influenza virus mutants (Hirst, I973). Isolation of recombinant virus and analysis of RNA Wallal mutant ts Io~ and Mudjinbarry mutant ts 3 were inoculated at low multiplicities of infection in mixed culture at 32 C and at cytopathic effect, the progeny were plated at 37 C. Sixty well-isolated plaques were selected and ground in ~ ml of medium ~99- Cultures of IO 6 cells were inoculated with o'5 ml of each plaque and grown at 32 C until cytopathic

5 Orbivirus recombinants 337 Table 2. Titre of temperature-sensitive mutants assayed at permissive and non-permissive temperatures Logto p.f.u./ml assayed at r EOP 37 C/32 C* Virus Mutant no. 32 C 37 C (- log10) Wallal 25 7"2 3' I 4'I 3o 6"4 1"7 4'7 34 5" ' "5 3'3 49 6' '7 66 7'6 3"0 4'6 73 5"5 2.I 3"4 8o 7-o 3'4 3'6 IOI 6"7 3"5 3.2 xo3 6'3 >7 4'6 IO9 6"4 3'6 2.8 Mudjinbarry 3 7' 3 4" 8 2' "1 3' I'7 4"8 I5 7-I 3"z 3'9 * Efficiency of plating at 37 C relative to 3z C. Table 3. Recombination frequencies between temperature-sensitive mutants of Wallal and Mudjinbarry viruses Group I: Wallal mutant ts 3o Group II: Wallal mutant ts IoI Mutant Group I Mudjinbarry ts3 ts I5 Wallal ts 25 ts 66 ts 80 ts 30 Group II zo p.f.u/cell o.oz p.f.u/cell 20 p.f.u/cell* 0.02 p.f.u/cell o o 3? 7 o o 3 3 o o 7 1 o o I 4 o o 8 23 o o I5 7 Mudjinbarry ts o Wallal ts lot I5 30 o * Combined multiplicity of infection of two mutants. t Recombination frequency defined in methods. effect. The whole of each culture was extracted for RNA and the double stranded (ds) RNA fraction separated by PAGE on slab gels of polyacrylamide together with RNA of parent viruses to detect aberrant RNA patterns. The gels were stained with ethidium bromide and three cultures produced RNA patterns which could not be reconciled with those of the parent viruses. RNA from plaque 27 showed an aberrant pattern in that segment 5 appeared to be derived from Mudjinbarry virus while, of the other segments clearly distinguishable between the parent viruses, segments 2 and IO appeared to be derived from Wallal virus. RNA from plaque 7 showed an aberrant pattern at segment 6 and RNA from plaque z8 at segment Io. The three plaques and six other randomly selected plaque isolates were assayed at 32 and 37 C and found not to be temperature sensitive.

6 338 B. M. GORMAN AND OTHERS ;:: ~i!!: ::; I ' i : : : ;z ':'./ij: ~i (a) (c) (b) (d) (e)... Fig 2. Separation of dsrna genome segments of (a) Wallal virus ts ~oi ; (b) a mixture of Wallal ts mutant IOI and recombinant 27; (c) recombinant 27; (d) a mixture of Mudjinbarry ts mutant 3 and recombinant 27; (e) Mudjinbarry ts mutant 3. Electrophoresis was performed in a downward direction in a gel slab of 7"5 % polyacrylamide and RNA stained with methylene blue (Gorman et al. I977). The isolates were plaque cloned and larger quantities of dsrna prepared for more detailed analysis. Fig. 2 shows the electrophoretic pattern of RNA of plaque 27 (recombinant 27) (Fig. 2 c) and co-electrophoresis of RNA of recombinant 27 with Wallal virus RNA (Fig. 2b) and with Mudjinbarry virus RNA (Fig. 2d). In the segments which can be distinguished between the viruses by co-electrophoresis, the recombinant virus appears to have derived segments 2, 3, 4, 6 and Io from Wallal virus and segment 5 from Mudjinbarry virus. The slight differences in mol. wt. make precise allocation of segment 4 difficult to establish and the origin of segments I, 7, 8 and 9 cannot be determined by this method. The electrophoretic pattern of RNA from recombinant 7 (Fig. 3 c) compared with RNA of Wallal virus (Fig. 3 b) and of Mudjinbarry virus (Fig. 3 d), showed that the recombinant

7 Orbivirus recombinants 339 W5 j a q R4. M4,,R5 M5 ~"~ R6, M6 (a) (b) (c) (at) (e) Fig. 3. Separation of dsrna genome segments of (a) mixture of recombinant 7 and Wallal ts mutant IOI; (b) Wallal ts mutant 1oi; (c) recombinant 7; (d) Mudjinbarry ts mutant 3; (e) mixture of recombinant 7 and Mudjinbarry ts mutant 3. Electrophoresis was performed in a downward direction in a gel slab of 7"5 % polyacrylamide and RNA stained with methylene blue (Gorman et al. I977). differed from the parent viruses in segments 4 and 6. Co-electrophoresis of RNA from the recombinant and Wallal virus (Fig. 3 a) suggests that segments 4 and 6 were derived from Mudjinbarry virus. This was confirmed in the co-electrophoresis of RNA of the recombinant and Mudjinbarry virus (Fig. 3 e) in which segments 4 and 6 of the viruses co-migrate. The recombinant appears to have derived segments 2, 3, 5 and Io from Wallal virus, segments 4 and 6 from Mudjinbarry virus with segments i, 7, 8 and 9 of undetermined origin. The electrophoretic pattern of RNA derived from plaque 28 differed from both parent viruses but on more detailed analysis two segments were found migrating near position Io (Fig. 4a). Attempts to isolate plaques with RNA segments including the larger tool. wt. segment Io, were unsuccessful. That the original material contained mixed virus populations was suggested by the isolation of two different RNA patterns in plaques selected from this material, The RNA patterns of two plaques (Fig. 4b and c) appeared similar to that of the parent Wallal virus while two other plaques (Fig. 4 d, e) showed similar patterns to that of recombinant 27 virus.

8 340 B. M. GORMAN AND OTHERS (a) (b) (c) (d) (e) Fig. 4. Separation of dsrna genome segments of (a) plaque isolate 28, (b) isolate 28 plaque I; (c) isolate 28 plaque 2; (d) isolate 28 plaque 3 ; (e) isolate 28 plaque 4. Electrophoresis was performed in a downward direction in a gel slab of 7"5 % polyacrylamide and the RNA stained with methylene blue (Gorman et al 1977). DISCUSSION The electrophoretic separation of the genome segments of Wallal and Mudjinbarry viruses revealed considerable differences between closely related viruses. Differences in the sizes of genome segments of the serotypes of the Warrego group of orbiviruses (Gorman et al. 1977) and between two serotypes of the Eubenangee group of orbiviruses (Gorman & Taylor, 1978) have been described. Heterogeneity in the sizes of genome segments has been reported for reovirus serotypes (Shatkin et al I968; Ramig et al 1977) and for isolates of cytoplasmic polyhedrosis virus (Payne & Rivers, I976 ). Extensive variation occurs between untyped isolates within the Wallal virus group (B. M. Gorman, J. Taylor and P. R. Young, unpublished data). The use of PAGE in analysis of RNA of plaques produced by mixed infections of temperature-sensitive mutants provides a method of determining the contribution of each parent virus to the genetic composition of recombinant viruses. The isolation of temperature-sensitive mutants and determination of recombination

9 Orbivirus recombinants groups of mutants was aimed at isolation of recombinants between viruses and the results are not intended as a study of rates of isolation of mutants nor aimed at the establishment of presumably ten recombinant groups corresponding to lesions in each of the ten genome segments. Isolation of eleven stable mutants from 125 non-enlarging plaques of Wallal virus and four stable mutants from I x3 non-enlarging plaques of Mudjinbarry virus need not be a measure of the effectiveness of 5-fluorouracil as a mutagen but probably reflects the method of selection of mutants. Ikegami & Gomatos 0968) suggested that the mutants of reovirus type 3 which they isolated after growth of virus in 5-fluorouracil might have arisen spontaneously. High frequency recombination between certain mutants and no recombination between other mutants (Table 3) was interpreted as indicating a mechanism of reassortment of genome segments rather than classical recombination. Similar results have been reported in recombination tests with temperature-sensitive mutants of reovirus (Fields, I97~) with temperature-sensitive mutants of influenza virus (Suguira, I975) and with temperaturesensitive mutants of bluetongue virus type Io (Shipham & De la Rey, I976). Independent reassortment of unlinked genome segments between temperature-sensitive viruses should yield a maximum recombination frequency of 25 % but in only a few cases (Table 3) did the established frequency reach the theoretical maximum. Recombination frequency was variable from experiment to experiment using the same pair of mutants in comparable experimental situations and similar variation has been reported in recombination tests using temperature-sensitive mutants of influenza virus (Suguira, ~975), of reovirus (Fields, I97I) and of bluetongue virus (Shipham & De La Rey, I976). Recombination appeared to be independent of input multiplicity (Table 3) and similar observations have been reported for influenza virus recombination (Hirst, I973). The profiles derived by PAGE of dsrna from parent and recombinant viruses (Fig. 2, 3) showed that genome segments of recombinant viruses were derived from both parents and provide further evidence that high frequency recombination observed between viruses with segmented genomes is due to independent reassortment of unlinked segments. In theory, recombinants involving each of the ten parental genome segments are possible, but only 256 recombinants can be expressed in a cross between two temperature-sensitive mutants. The detection system based on comparative mobilities of RNA isolated from different plaques cannot detect all possible recombinants. The method of plaque isolation would ensure that only recombinants in highest yield were selected. The identification of recombinants by screening RNA patterns in PAGE allows for detection of recombinants in only three or four segments. However, the use of PAGE to demonstrate interchange of RNA segments between orbiviruses confirms similar findings with influenza viruses (Palese, I977) and with reoviruses (Sharpe et al. t978 ). The transfer of RNA segments from one orbivirus serotype to another has implications for other orbivirus groups. Antigenic diversity within orbivirus serological groups has been described, particularly with the important bluetongue virus group where the plurality of strains has long been recognized (Howell, I966 ). Reassortment of genome segments coding for antigenic properties or virulence determinants between bluetongue virus strains or perhaps between bluetongue virus and other orbiviruses, could be an important mechanism in the evolution of bluetongue virus strains. Attempts to isolate recombinants between serologically unrelated orbiviruses are in progress. This work was supported by a grant from the National Health and Medical Research Council, Canberra. We thank Mr A. J. Melzer for technical assistance. 34t

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